Orientador: Ricardo Utsunomia / Resumo: Pesticidas organofosforados são muito utilizados na agricultura, mas são tóxicos para muitos organismos incluindo os seres humanos, podendo atuar como desreguladores endócrinos e como agentes mutagênicos e carcinogênicos. Um dos efeitos desreguladores é a alteração do metabolismo lipídico, que está associado também ao desenvolvimento do câncer. Isso pode ser explicado pela necessidade das células tumorais em utilizar ácidos graxos para compor suas membranas em construção. Dentre os pesticidas organofosforados está o Diclorvós (DDVP), cujo alto consumo e utilização se baseia em sua grande eficácia e baixo custo. Neste sentido, o objetivo do presente estudo foi avaliar a influência do praguicida DDVP sobre os marcadores moleculares do metabolismo lipídico (SREBP, SCAP, LIMP-II e CD36) na próstata de ratos após indução química pelo carcinógeno N-metil-N-nitrosoureia (MNU). Foram utilizados 32 ratos da linhagem Fischer 344, com idade de 90 dias. Os ratos foram separados em quatro grupos experimentais: Controle, DDVP, MNU, MNU+DDVP. Foram feitas análises histopatológicas, imuno-histoquímicas e Western Blotting na próstata ventral dos ratos. Na análise histopatológica, os grupos MNU e MNU+DDVP apresentaram 100% de incidência de hiperplasia. Para a avaliação morfométrico-estereológica, o grupo MNU+DDVP apresentou aumento do volume relativo de epitélio quando comparado com o grupo controle. As proteínas SREBP e CD36 foram encontradas nas células epiteliais luminais na região do Apare... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Organophosphate pesticides are widely used in agriculture, but are toxic to many organisms including humans, and can act as endocrine disrupters and as mutagenic and carcinogenic agents. One of the deregulatory effects is the alteration of lipid metabolism, which is also associated with cancer development. This may be explained by the need for tumor cells to use fatty acids to compose their building membranes. Among the organophosphate pesticides, Dichlorvos (DDVP) is one of the most effective and least expensive. Therefore, the objective of the present study was to evaluate the influence of the DDVP pesticide on the molecular markers of lipid metabolism (SREBP, SCAP, LIMP-II and CD36) in rat prostate after chemical induction by the carcinogen N-methyl-N-nitrosourea (MNU). Thirty two rats from the Fischer 344 lineage aged 90 days were used. The rats were separated into four experimental groups: Control, DDVP, MNU and MNU + DDVP. Histopathological, immunohistochemical and Western Blotting analyzes of the ventral prostate of rats were performed. In the histopathological analysis, the MNU and MNU + DDVP groups had a 100% incidence of hyperplasia. For the morphometric-stereological evaluation, the MNU + DDVP group showed an increase in the relative volume of epithelium when compared with the control group. The SREBP and CD36 proteins were found in luminal epithelial cells in the Golgi Apparatus region, SCAP mainly in the apical region, and LIMP II dispersed in the cytoplasm as cl... (Complete abstract click electronic access below) / Mestre

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation of the epidermal growth factor receptor (EGFR). As a result, the cellular response to EGF was reduced as shown by the down-regulation of EGF-stimulated Erk1/2 phosphorylation. The expression of Slug, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and granulocyte-macrophage colony stimulating factor (GM-CSF) was also down-regulated by prostasin re-expression in the PC-3 cells. Co-expression of prostasin and its activating protease matriptase with EGFR in FT-293 cells induces an apparent proteolytic cleavage of the EGFR in the extracellular domain at two specific sites, generating two N-terminally truncated EGFR fragments, named EGFR135 and EGFR110. The EGFR110 is constitutively tyrosine-phosphorylated, and in its presence the phosphorylation of downstream signaling molecules including Erk1/2 and Akt is increased under serum-free conditions. Neither EGFR135 nor EGFR110 is responsive to EGF stimulation. Deletions of the EGFR extracellular domain (ECD) were generated to map the matriptase-prostasin cleavage sites. Two candidate sites were localized to regions AA1-273 and AA273-410. These data support a mechanism of action for the matriptase-prostasin epithelial extracellular serine protease activation cascade by proteolytically modulating the EGF-EGFR signaling. Prostasin gene expression is down-regulated in high-grade and hormone-refractory prostate cancers. We investigated the mechanisms by which androgens regulate prostasin expression in the prostate and prostate cancer. We treated the LNCaP human prostate cancer cells with dihydrotestosterone (DHT) and measured the mRNA expression of prostasin and potential transcription regulators of prostasin predicted by interrogation of the prostasin gene promoter sequence. Prostasin mRNA expression in the LNCaP cells was not responsive to DHT treatment. DHT marginally up-regulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of a prostasin promoter-reporter and SREBP cDNA in HEK-293 cells resulted in stimulation of the promoter activity at ~2 fold by SREBP-1c, and up to 6 fold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of the promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2 s stimulation of the prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 cells resulted in up-regulation of the endogenously expressed prostasin while transfection of a SLUG cDNA in the LNCaP cells repressed prostasin expression. Multiple SREBP-2 binding sites, known as sterol regulatory elements (SRE s), were identified at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Mutagenesis of the five SRE s was carried out to evaluate their roles in SREBP-2 up-regulation of prostasin. SRE+98, a novel functional sterol regulatory element was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. CONCLUSIONS: Prostasin regulates the expression of several invasion-promoting molecules in prostate cancer cells by down-modulating the EGF-EGFR signaling pathway. Active prostasin induces proteolytic cleavage in the EGFR ECD at two specific sites. One of the N-terminally truncated EGFR, the EGFR110 is auto-phosphorylated along with increased phosphorylation of downstream signaling molecules. The effect of the androgen DHT on prostasin expression in prostate cells is mediated via SREBP s, which stimulate the promoter, and Slug, which represses the promoter. Slug is up-regulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

<p> Cellular cholesterol homeostasis is a fundamental and highly regulated process. Transcription factors known as sterol regulatory element binding proteins (SREBP) are responsible for the expression of many genes involved in the uptake and biosynthesis of cholesterol. SREBP activation and lipid dysregulation has been associated with cellular endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). Our lab has previously reported a relationship between ER stress and SREBP activation causing lipid dysregulation and hepatic steatosis. This project was designed to elucidate the mechanism of ER stress-induced SREBP activation and determine its relationship with cellular pathologies associated with ER stress and lipid accumulation. My research has examined the mechanism by which ER stress activates SREBP-2 in various cell lines, including epithelial and macrophage cells. This research revealed that (1) ER stress-induced SREBP-2 activation is not dependent on caspases and occurs through the conventional sterol-mediated proteolytic pathway; (2) the mechanism of ER stress-induced SREBP-2 activation is sensitive to changes in ER calcium; (3) ER stress is associated with SREBP-2 activation and lipid dysregulation in a model of renal injury; and ( 4) ER stress-induced SREBP activation in vitro is not associated with lipid accumulation in macrophage foam cells. </P> <p> This project has also offered me the opportunity to further enhance our understanding of the mechanism by which ER stress causes SREBP activation in a sterolindependent manner. </P> / Thesis / Doctor of Philosophy (PhD)

<p>End stage renal disease (ESRD) has been a world-wide cause of renal disease-related fatality. Most patients with ESRD progress through chronic kidney disease (CKD). There are various causes of CKD including mechanical stress, infections, high blood pressure or immune conditions. Renal fibrosis is known to play an important pathogenic role in CKD and ESRD. Transforming growth factor-beta (TGF-ß) is one of the major mediators of fibrosis, and inhibition of TGF-ß could reduce renal fibrosis in in vitro models. One of the well - known inducers of TGF-ß transcr iption is Angiotensin II (ANGII). 1 This is a major contributor to the matrix accumulation and glomerular fibrosis leading to a decline in kidney function and kidney failure in patients with chronic kidney disease (CKD). 2 A recent study demonstrated that TGF -ß could be increased by SREBP -1c overexpression. 3 My laboratory’ s data showed that ANGII could induce TGF-ß on a transcriptional level, and this induction could be block ed by Fatostatin, an SREBP/SCAP inhibitor. My data showed that ANGII could induce SREBP-1 in a time and dose dependent manner. Therefore my study focused on understanding the role of SREBP-1 in ANGII-induced TGF-ß upregulation in primary mesangial cells (MCs). We found that ANGII- induced TGF-ß promoter activity requires Angiotensin type 1 receptor (AT1R) and SREBP-1 activation as well as ER stress. Interestingly , the activation of SREBP-1 by ANGII stimulation relies on ER stress and PI3K/AKT signaling. Both ANGII and ER stress inducer s could induce AKT phosphorylation, indicat ing that ER stress is an upstream mediator in th is signaling pathway. We also found that ER stress inhibit ion could block SREBP-1 activation by ANGII stimulation and, more importantly , could also block ANGII- induced TGF-ß promoter activity. In C57BL/6 mice infused with ANGII for one week, we found that both SREBP-1 and GRP78 were significantly upregulated in glomeruli in the ANGII infusion group compared with the control group. Taken together , this data indicates that ER stress is required for SREBP-1 activation, both of which are mediators of kidney fibrosis.</p> / Master of Science (MSc)

<p>Cytochrome b5 heme binding domain 2 (CYB5D2) is a heme binding protein that was initially identified for its ability to attenuate the function of the PTEN tumor suppressor gene. CYB5D2 sustains ectopic PTEN expression in U87 cells, and can also confer survival from serum starvation in NIH3T3 cells. An antibody was generated to the carboxyl terminus of CYB5D2 to detect endogenous protein expression. The highest expression of CYB5D2 protein is in neural cancer cell lines. CYB5D2 is weakly expressed in breast and kidney cancer cell lines, and moderately expressed in prostate cancer cell lines. To investigate the role of the heme binding domain in CYB5D2, a conserved aspartic acid (D86) within this domain was mutated to glycine, and this was characterized as being unable to bind heme. CYB5D2(D86G) displayed a loss of function compared to wild-type CYB5D2. To study the loss of expression of CYB5D2, stable CYB5D2 shRNA was achieved in HeLa and Huh7 cells. While ectopic CYB5D2 inhibited HeLa cell proliferation and growth in soft agar, CYB5D2(D86G) expression and CYB5D2 shRNA increased cell proliferation and soft agar growth. While ectopic CYB5D2 conferred survival from chemotherapeutic drugs in HeLa cells, CYB5D2(D86G) and CYB5D2 shRNA cells were susceptible to drug treatments. CYB5D2 inhibits SREBP signalling, which requires its heme binding ability. Using cyclohexamide treatments, CYB5D2 stabilized ectopic Insig1, while CYB5D2(D86G) destabilized ectopic Insig1. CYB5D2 shRNA reduced endogeneous CYP51A1 (lanosterol demethylase) and Insig1 protein levels, and increased the susceptibility of HeLa cells to mevalonate treatments. Furthermore, CYB5D2 shRNA HeLa cells displayed reduced CYP3A4 activity, a cytochrome P450 enzyme involved in drug metabolism. CYB5D2 binds to cytochrome P450 reductase (POR), while CYB5D2(D86G) cannot. CYB5D2 co-immunoprecipitates with endogenous POR under serum-free conditions in HeLa and Huh7 cells, while CYB5D2(D86G) cannot. Collectively, CYB5D2 is a POR interacting protein, which regulates CYP51A1 and CYP3A4 activity.</p> / Doctor of Philosophy (Medical Science)

<p>There has been a steady increase in the number of patients with chronic kidney disease. The etiology has been linked to excessive fibrosis progression until the kidney function becomes compromised. We are investigating tubulointerstitial fibrosis (TIF) specifically, as it correlates strongly with the decline of renal function.</p> <p>In our study we investigated the role that active SREBP may play in apoptosis and fibrosis in vivo and in vitro. Treating HK-2 cells with TNFα resulted in the cleavage of SREBP and activation of its SRE promoter. By utilizing a number of inhibitors, we found TNFα induced SREBP cleavage through both a caspase independent and dependent manner.</p> <p>We used fatostatin, a SCAP inhibitor, to reduce the amount of active SREBP in animals in the unilateral ureter obstruction (UUO) model. This model is well known for its development of TIF. Fatostatin decreased SREBP-1 and SREBP-2 activation in mice after 7 and 14 days. Fatostatin increased glomerulotubular integrity and proximal tubular mass as evaluated using lectin staining, along with reducing the number of cells undergoing apoptosis as evaluated by TUNEL staining. Using the Masson Trichrome, Picrosirius red and fibronectin staining, we found a reduction of fibrosis. Fatostatin was also found to attenuate the accumulation of infiltrating myofibroblasts and T cells. These results point to a pathological role for SREBP in TIF.</p> / Master of Health Sciences (MSc)

Chez le nématode Caenorhabditis elegans (C. elegans), le cofacteur mdt-15, orthologue à Med15 chez le mammifère, est essentiel à l’homéostasie des lipides. Aussi, l’inhibition pharmacologique de l’interaction entre Med15 et le facteur de transcription SREBP améliore le profil lipidique chez des souris obèses. Le foie, organe important du métabolisme énergétique, peut subir des dérèglements lors du vieillissement et dans l’obésité. La modulation des niveaux hépatiques de Med15 dans ces conditions pathologiques est toutefois inconnue. Cette étude visait donc à évaluer l’expression de Med15 au foie dans le vieillissement et l’obésité. Les modèles de vieillissement étaient des souris C57Black/6J (B6), des rats Sprague-Dawley (SD) et des rats Lou (modèle de vieillissement réussi) de différents groupes d’âges et sous diète faible en gras (LFD). MED15 a également été mesuré dans du foie humain provenant de 3 groupes de patients obèses jeunes, d’âge intermédiaire et vieux. Afin de dissocier les effets du vieillissement de ceux de l’obésité, Med15 a été mesuré dans des souris ob/ob ou db/db sous LFD et des souris B6 sous diète riche en gras (HFD) âgées de 4 mois. L’expression de Med15 était diminuée chez les souris B6 et les rats SD âgés mais demeurait stable chez les rats Lou vieux et les patients âgés. Les niveaux de Med15 étaient diminués chez les souris ob/ob et db/db. En revanche, ses niveaux protéiques hépatiques étaient augmentés chez les souris sous HFD. La conclusion générale, tirée des liens établis entre les résultats présentés ici et la littérature, est que l’expression de Med15 serait bénéfique chez un organisme sain mais que sa diminution permettrait de freiner les troubles métaboliques associés au vieillissement et à l’obésité. Des études in vitro et in vivo sur les impacts des variations observées dans cette étude permettraient de caractériser Med15 comme modulateur métabolique chez le mammifère. / In the nematode Caenorhabditis elegans (C. elegans), the mdt-15 cofactor, orthologous to the mammalian Med15, is essential for lipid homeostasis. Furthermore, pharmacological inhibition of the interaction between Med15 and Sterol Regulatory Element Binding Protein (SREBP) transcription factor improves the lipid profile in obese mice. The liver, important organ of energy metabolism, may undergo disorders during aging and in obesity. Modulation of Med15 hepatic levels under these two conditions is however unknown. This study aimed therefore to evaluate hepatic Med15 expression in several aging and obesity models. The aging models were C57Black/6 Jackson (B6) mice, Sprague-Dawley (SD) rats and Lou rats (a successful aging model) in different age groups and under low-fat diet (LFD). MED15 was also measured in human liver from 3 groups of obese young, middle-aged and old patients. In order to dissociate the effects of aging from those of obesity, Med15 expression was measured in 4 months old ob/ob or db/db mice under LFD and B6 mice under high-fat diet (HFD). Med15 expression was decreased in old B6 mice and SD rats but remained stable in old Lou rats and elderly patients. Med15 levels were diminished in ob/ob and db/db mice. However, Med15 protein levels were increased in mice under HFD. The general conclusion, drawn from links established between the results presented here and the literature, is that Med15 expression would be beneficial in a healthy organism but its decrease would curb the metabolic disorders associated with aging and obesity. In vitro and in vivo studies on the impacts of the variations observed in this study would allow for the Med15 characterization as a key metabolic modulator in mammals. / Résumé en espagnol

Les données de la littérature rapportent la supériorité de la chirurgie bariatrique sur le traitement médical optimisé concernant la perte pondérale et l'amélioration du diabète de type 2. Les facteurs prédictifs de bons résultats en terme pondéral et métabolique restent encore méconnus et des échecs sont constatés. Le phénotypage de l'obésité et de son retentissement métabolique semble essentiel afin d'adapter la procédure chirurgicale au cas par cas et améliorer les résultats. Dans ce travail de thèse, par une approche clinique, nous avons cherché à identifier les facteurs prédictifs d'amélioration des paramètres métaboliques et de succès pondéral après chirurgie bariatrique. Nous avons démontré le rôle majeur de la perte de poids après chirurgie dans l'amélioration du métabolisme glucidique et des paramètres métaboliques. Nous avons également montré l'impact positif de la masse musculaire initiale sur la perte pondérale, facteur également déterminant dans le contrôle du métabolisme glucidique. Les marqueurs du dysfonctionnement cellulaire Beta sont également apparus déterminants pour prédire la rémission du diabète de type 2 après chirurgie. Ainsi, l'efficacité de la chirurgie dans le contrôle du syndrome métabolique, au-delà de la technique opératoire, apparaît très dépendante de la perte de poids mais aussi du terrain, confirmant l'importance du phénotypage de l'obésité en préopératoire. Par une approche expérimentale, nous avons cherché à identifier l'impact du tissu adipeux sur les organes sièges de l'insulino-résistance (muscle et foie) impliqués dans le syndrome métabolique. La constitution de la tissuthèque DioMede et l'obtention de milieux conditionnés de tissu adipeux nous ont permis d'étudier l'impact des sécrétions de ce tissu sur les tissus insulino-sensibles en se rapprochant des conditions physiologiques. Nous avons identifié un effet direct du tissu adipeux sur le métabolisme musculaire des acides gras (AG) par la régulation négative du facteur de transcription SREBP-1c. Nos résultats identifient les acides gras insaturés comme les médiateurs de l'inhibition de SREBP-1, conduisant à une diminution de la lipogenèse par l'intermédiaire des gènes cibles de ce facteur de transcription. La composition et les proportions respectives d'AG mono ou poly insaturés et d'AG saturés dans le tissu adipeux, leur niveau de sécrétion, et leur taux circulant apparaissent donc déterminants dans la régulation de la lipogenèse des tissus insulino-sensibles (foie et muscle), et pourraient être un marqueur des obésités avec désordres métaboliques / Literature data reported the superiority of bariatric surgery on optimized medical treatment concerning weight loss outcomes and improvement of type 2 diabetes. Predictive factors of good weight loss results and metabolic control are still unrecognized and failures are recorded. Phenotyping obesity and its metabolic consequences seem essential to tailor the surgical procedure to each patient and to improve the outcomes. In this work, by a clinical approach, we have tried to identify predictive factors of metabolic control and weight loss after bariatric surgery. We have demonstrated the major role of weight loss to achieve glucose homeostasis and metabolic control. We have also reported the positive impact of initial Fat Free Mass on weight loss outcomes and glucose metabolism control. Beta cell dysfunction markers appeared to also have a major impact on Type 2 Diabetes remission after surgery. Thus, the efficacy of surgery on metabolic control, beyond the surgical technique, seems highly related to weight loss and patients history, which underlines the importance of phenotyping obesity before surgery. By an experimental approach, we have tried to identify the impact of adipose tissue on muscle and liver, organs that are involved in the metabolic syndrome. By means of a tissue collection (Diomede) and the use of conditioned media of adipose tissue, we studied the impact of adipose tissue secretions on insulin sensitive tissues, close to physiological conditions. We found a direct effect of adipose tissue on fatty acid metabolism in muscle through SREBP-1c down regulation. Unsaturated Fatty Acids were identified as the mediators of SREBP-1 inhibition, leading to a decrease in lipogenesis through target genes of this transcriptional factor. The composition and the respective proportion of mono or poly unsaturated fatty acids and saturated fatty acids in adipose tissue, their level of secretion, and their circulation rate appear to be determinant in lipogenesis regulation in insuline sensitive tissues (muscle and liver), and could be markers of metabolic disorders in obese patients

La proprotéine convertase subtilisine/kexine type 9 (PCSK9) favorise la dégradation post-transcriptionnelle du récepteur des lipoprotéines de faible densité (LDLr) dans les hépatocytes et augmente le LDL-cholestérol dans le plasma. Cependant, il n’est pas clair si la PCSK9 joue un rôle dans l’intestin. Dans cette étude, nous caractérisons les variations de la PCSK9 et du LDLr dans les cellules Caco-2/15 différentiées en fonction d’une variété d’effecteurs potentiels. Le cholestérol (100 µM) lié à l’albumine ou présenté en micelles a réduit de façon significative l’expression génique (30%, p<0,05) et l’expression protéique (50%, p<0,05) de la PCSK9. Étonnamment, une diminution similaire dans le LDLr protéique a été enregistrée (45%, p<0,05). Les cellules traitées avec le 25-hydroxycholestérol (50 µM) présentent également des réductions significatives dans l’ARNm (37%, p<0,01) et la protéine (75%, p<0,001) de la PCSK9. Une baisse des expressions génique (30%, p<0,05) et protéique (57%, p<0,01) a également été constatée dans le LDLr. Des diminutions ont aussi été observées pour la HMG CoA réductase et la protéine liant l’élément de réponse aux stérols SREBP-2. Il a été démontré que le SREBP-2 peut activer transcriptionnellement la PCSK9 par le biais de la liaison de SREBP-2 à son élément de réponse aux stérols situé dans la région proximale du promoteur de la PCSK9. Inversement, la déplétion du contenu cellulaire en cholestérol par l’hydroxypropyl-β-cyclodextrine a augmenté l’expression génique de la PCSK9 (20%, p<0,05) et son contenu protéique (540%, p<0,001), en parallèle avec les niveaux protéiques de SREBP-2. L’ajout des acides biliaires taurocholate et déoxycholate dans le milieu apical des cellules intestinales Caco-2/15 a provoqué une baisse d’expression génique (30%, p<0,01) et une hausse d’expression protéique (43%, p<0,01) de la PCSK9 respectivement, probablement via la modulation du FXR (farnesoid X receptor). Ces données combinées semblent donc indiquer que la PCSK9 fonctionne comme un senseur de stérols dans le petit intestin. / Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 µM) solubilised in albumin or micelles significantly down-regulated PCSK9 gene (30%, p<0,05) and protein expression (50%, p<0,05), surprisingly in concert with a decrease in LDLr protein levels (45%, p<0,05). 25-hydroxycholesterol (50 µM) treated cells also displayed significant reduction in PCSK9 gene (37 %, p<0,01) and protein (75% p<0,001) expression, while LDLr showed a decrease at the gene (30%, p< 0,05) and protein (57%, p<0,01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of HMG-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-β-cyclodextrine up-regulated PCSK9 transcripts (20%, p<0,05) and protein mass (540%, p<0,001), in parallel with SREBP-2 protein levels. The addition of bile acids, taurocholate and deoxycholate, to the apical culture medium lowered PCSK9 gene expression (30%, p<0,01) and raised PCSK9 protein expression (43%, p<0,01) respectively, probably via the modulation of farnesoid X receptor. Combined, these data indicate that PCSK9 functions as a sensor of sterol in the small intestine.

Sirt1 (Sirtuine 1) est une protéine histone déacétylase dépendante de NAD+ qui stimule la néoglucogénèse et inhibe la glycolyse dans le foie, et qui augmente l'oxydation des acides gras dans le muscle strié squelettique. Le but de ce travail de thèse a été de définir les fonctions métaboliques de Sirt1 dans le muscle strié squelettique. Nous avons tout d'abord montré, à l'aide d'un modèle de souris déficientes pour le gène Sirt1, que Sirt1 régulait l'expression de l'hexokinase II et de SREBP-1c, protéine régulatrice de l'expression de l'hexokinase. De plus, un modèle d'électrotransfert de gènes permettait de mettre en évidence que Sirt1 régulait l'expression de SREBP-1c de façon LXR dépendante. Enfin, l'inhibition de Sirt1 par l'EX527 aboutissait à une diminution de la consommation de glucose chez des myotubes C2C12. Prises ensemble, ces données suggèrent un rôle important de Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique. Dans un second temps, nous avons déterminé le rôle potentiel de Sirt1 lors d'un jeûne chez des myotubes C2C12. Un jeûne entraînait une augmentation de l'activité cathepsine B + L et une déphosphorylation des protéines AktS473, GSK3S21/S9, p70S6KT412 et S6 S235/S236 qui précédait une amyotrophie des myotubes. La renutrition aboutissait à une rephosphorylation de ces protéines et à un retour à la normale de la taille des myotubes. L'activité cathepsine B + L restait cependant élevée. Enfin, le niveau en ARNm de Sirt1 était augmenté de façon transitoire lors de la renutrition. D'autres mesures de marqueurs des voies protéolytiques et de l'activité de Sirt1 sont à envisager. Nos données ainsi que celles de la littérature suggèrent que Sirt1 pourrait avoir un rôle dans la régulation de l'autophagie lors du jeûne. Pour conclure, ce travail de thèse met en évidence un rôle pour Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique et apporte de nouvelles perspectives dans l'étude de la régulation de ce métabolisme en conditions pathologiques